The diagnosis of a large number of medically relevant conditions is currently performed using molecular markers as tools. The molecular tools are generally used as one aspect in a complex examination, taking into account a series of different parameters characterizing the samples to be examined.
In medically relevant analysis, the morphological examination of samples by cytological or histological means is in common use. Such methods based on morphological characterization of cell based samples are applicable for example in analysis of clinical samples such as body fluids, blood, surgical resections, secretions, swabs or lavages.
In screening for cervical cancer, for example, swabs are used for detection of neoplastic lesions of the cervix uteri. In the screening procedure, lesions of different origin have to be distinguished. Causes for lesions may for example be inflammations (due to infectious agents or physical or chemical damage) or preneoplastic and neoplastic changes. In morphological examinations the lesions of different characteristics are sophisticated to distinguish. Thus, for examination of swabs, cytologists and pathologists have to be especially trained and even experienced examiners have a high inter- and intra-observer variance in the assessment of a diagnosis based on cytological specimens. In general the result of the examination is based upon the subjective interpretation of diagnostic criteria by the examining pathologist/cytologist. As a result the rate of false positive and false negative results in the screening tests remains unsatisfying high.
Therefore, in many cases these cytological or histological examination procedures are supported by the use of molecular markers. Such markers are often used in immuno-histochemical staining reactions, or in the course of in-situ hybridization reactions. In the prior art combinations of morphological examinations and immuno-histochemical staining reactions based on marker molecules, characteristic for different medically relevant states of tissues or cells, may lead to enhanced results. The morphologic examination remains laborious and time consuming and thus expensive, even when supported by the molecular methods, that make the results more reliable. Additionally, the diagnosis on a morphologically cell based level is, even when supported by molecular parameters, subject to individual perception of the morphology by individual examiners. Thus the diagnosis is dependent on the person, that performs the examinations.
Only in very few cases, molecular markers may be used as diagnostic tools without further support by cell based morphological examinations. This is especially the case, if markers are to be detected in an environment, where they do only occur under exactly defined conditions. So the methods for diagnosis of conditions on a molecular level only, without the support of cell based information, are restricted to cases, where there are suitable markers, that are non-ambiguously specific for the condition to be characterized. For example, detection of viral infections may be carried out in solutions of samples, because the markers characteristic for the presence of viruses in tissues do not occur in unaffected human tissues.
The reproducibility of the results of examination can be enhanced by the use of supporting molecular tools. However, the problem with the preservation and preparation of the samples may not be overcome by just additionally using molecular markers.
When using molecular tools in cytological or histological examinations, strict precautions in preserving the samples have to be taken to prevent artefacts and improper results of the tests. This is in part due to the instability of the cell based morphological information and in part to the instability of the molecular markers to be detected during the tests. If the samples are not prepared, transported or stored in the appropriate manner, the cell based information, or even the molecular information may get lost, or may be altered. So the diagnosis may be impossible, or may be prone to artefacts. For example, the interpretation of biopsies or cytological preparations is frequently made difficult or impossible because of damaged (physically or biochemically) cells. Regarding tissue samples or biopsies, the preservation of molecular constituents of the samples, which are subject to a rapid turnover, seems sophisticated due to the time elapsed until penetration of the total sample by appropriate preservatives.
The morphologically supported diagnostic methods performed routinely in the art show two major disadvantages. First, the methods are highly dependent on individual perception of the examiners. Secondly the morphological information is quite sensitive to decay processes and thus may cause artefacts after preparation of the samples. Both aspects contribute to improper reproducibility of the results.
For improved diagnosis of medically relevant conditions, methods that do not depend on cell based morphological information would be desirable.